During the past fiscal year, we have made substantial progress with three ongoing projects that include (i) identification, and biophysical and structural characterization of novel carbohydrate-binding proteins that inhibit HIV-1 entry; (ii) design of HIV?1 and HIV?2-derived gp41 peptide and protein analogs and evaluation of their properties as HIV entry inhibitors and as novel immunogens; and (iii) synthesis and evaluation of mycothiol-associated enzymes in Mycobacterium tuberculosis. (i) Earlier we identified a new cyanobacterial protein MVL that was originally isolated from a cultured cyanobacterial strain Microcystis viridis NIES102. Using a variety of biophysical techniques and NMR, we demonstrated that MVL binds with low micromolar affinity a minimal tetrasaccharide epitope known as Man-2A [Man-alpha (1-6) Man-beta (1-4) GlcNAc-beta (1-4) GlcNAc], and binds higher molecular weight oligomannosides such as Man-6 and Man-9, both of which contain the Man-2A core, with sub-micromolar affinities. Using NMR chemical shift perturbations, we mapped to the amino acid sequence two symmetry-related carbohydrate binding sites in MVL and unambiguously showed that MVL exists as a homodimer with internal symmetry. Further, MVL inhibits HIV-1 fusion as evaluated in both a fusion and single round infectivity assay against M- and T-tropic strains SF162 and LAV, respectively, with an approximate IC50 value of 30 nM in each assay. MVL?s inhibitory activity can be competed with high mannose oligosaccharides such as Man-8 and Man-9 indicating that MVL inhibits fusion through carbohydrate-mediated interactions. To establish the structural basis for MVL?s HIV-1 inhibitory activity and unprecedented fine carbohydrate specificity, in collaboration with David Williams and Jae Young, we have solved the crystal structures of MVL free and bound to the pentasaccharide Man3GlcNAc2 at 1.9 ? and 1.8 ? resolution, respectively. Consistent with our biophysical studies that established MVL as a homodimer in solution, the dimeric structure is stabilized by an extensive intermolecular interface between monomers. Each monomer contains two structurally homologous domains with high sequence similarity connected by a short five amino acid residue linker. Residual dipolar coupling measurements indicate that the structure of the MVL dimer in solution is identical to that in the crystal. Man3GlcNAc2 binds to a preformed Y-shaped cleft at the distal end of each domain such that a total of four independent carbohydrate molecules associate with each homodimer. The binding cleft provides shape complementarity to the Y-shape of the branching ligand Man3GlcNAc2 and includes the presence of a deep hydrophobic hole that accommodates the N-acetyl methyl at the reducing end of the carbohydrate. Specificity arises from seven to eight intermolecular hydrogen bonds, and a Gln side chain projects from the binding site to perfectly accommodate the branch point of the high mannose oligosaccharides containing the Man3GlcNAc2 core. (ii) Recall the goals of this project are 3-fold and include designing new fusion inhibitors with nM IC50s, designing inhibitors that can act as probes to better define the precise temporal and structural steps leading to fusion, and constructing stable N-helical trimers that will elicit neutralizing antibodies. In collaboration with Robert Blumenthal?s laboratory and using our designed peptide and protein gp41 analogs, during FY 2005 we demonstrated that the monoclonal antibody NC-1, which was raised against the 6-helix bundle (6-HB) core of the ectodomain of gp41, in fact is highly specific for our covalently linked N-helical trimers, in addition to 6-HB. This result indicates that NC-1 recognizes a portion of the N-peptide trimer that is exposed in the structure of 6-HB. In terms of elicitation and panning of anti-gp41 antibodies, work begun in collaboration with two different groups (Zwick, Burton and Mage, TSRI and NIAID, and Antibodies by Design, GmbH) have resulted in selection of a series of Fabs with different cross-reactivities. For example, antibodies specific for the N-trimer, for 6-HB and for both have been identified and characterized. We have shown that two of these modestly inhibit HIV-1 fusion in a fusion assay (with IC50 values in the range of 10 micrograms/mL), but only weakly inhibit infectivity in a neutralization assay. Further experiments are underway to optimize affinity of these antibodies toward pre-hairpin intermediates of gp41 with the intent of increasing neutralization potency, as well as identifying additional new antibodies from Mage?s rabbits that were immunized over extended periods with our various N-trimer constructs. This work will continue through FY 2006. (iii) Last, we have continued efforts toward synthesizing small molecule inhibitors of mycothiol-associated enzymes, paying particular attention to the biosynthetic enzyme GlcNAc-Ins deacetylase and the detoxification enzyme mycothiol-S-conjugate amidase (MCA), both of which are metalloenzymes. Our strategy has been to design and synthesize small molecules that incorporate features of a series of bromotyrosine-derived natural products that we showed earlier to inhibit MCA, as well as to preserve half of the features of the natural substrate, namely mycothiol-conjugates, for docking and binding to the enzyme?s active site. We have completed the synthesis of two classes of inhibitors that are built upon wither a quinic acid-glucosamine scaffold or a cyclohexyl-S-glucosamine scaffold. These compounds are currently being evaluated for their inhibitory properties toward these enzymes. Last, on the basis of preliminary results and in an attempt to simplify the synthetic schemes employed to obtain the above mentioned libraries, we have synthesized several simpler analogs that are built upon glucosamine alone, that is we have dispensed with the inositol-replacement in this final series of compounds. The biological assay results will guide future synthetic efforts.